CN114436933A

CN114436933A - Vitamin B6Preparation method and application of impurity compound

Info

Publication number: CN114436933A
Application number: CN202210358355.2A
Authority: CN
Inventors: 贾慧娟; 任晓慧; 侯伟; 李世奇
Original assignee: Beijing Creatron Institute Of Pharmaceutical Research Co ltd; Tianjin Ruichuang Kangtai Biotechnology Co ltd
Current assignee: Beijing Creatron Institute Of Pharmaceutical Research Co ltd; Tianjin Ruichuang Kangtai Biotechnology Co ltd
Priority date: 2022-04-07
Filing date: 2022-04-07
Publication date: 2022-05-06

Abstract

The invention provides vitamin B₆The preparation, separation and purification method of the reference substance for degrading impurities can prepare high-purity B rapidly and massively at low cost₆Degrading impurity reference substance, solving the problem of vitamin B₆The quality control of raw materials and preparations thereof and the lack of impurity reference substances in the research of impurities. For further improving vitamin B₆And the quality safety of the preparation product thereof, the physical health of the consumers and the like are providedThe technical support of (2) has important practical significance.

Description

Vitamin B6Preparation method and application of impurity compound

Technical Field

The invention relates to chemical pharmacyThe field of the technology. More particularly, the present invention relates to vitamin B₆The preparation, standardization and structure identification of impurities in the bulk drugs and the products on the market, and the application of the bulk drugs and the products on the market as impurity reference substances.

Background

Vitamin B₆Also known as pyridoxine. The name of English: vitamin B₆(abbreviation: VB)₆) Chinese culture name: 6-methyl-5-hydroxy-3, 4-pyridinedimethanol hydrochloride having the following formula (I):

vitamin B₆As a water-soluble vitamin, it is contained in yeast, liver, grain, meat, fish, egg, bean and peanut in a large amount. Vitamin B₆Is a component of coenzyme in human body, participates in various metabolic reactions, and especially has close relation with amino acid metabolism. Vitamin B₆The injection was marketed in Japan as early as 1957, and was standardized at 1ml:10mg and 1ml:30 mg. The clinical indications include the use of vitamin B₆Prevention and treatment of deficiency; preventing and treating isoniazid poisoning; can be used for treating emesis caused by pregnancy, radiation sickness and anticancer drugs; seborrheic dermatitis, etc. Vitamin B₆The injection can also be used for total parenteral nutrition, and vitamin B in case of malnutrition and progressive weight loss due to insufficient intake₆And (4) supplementing. In addition, the following is true for vitamin B₆The required amount increases: pregnancy and lactation, hyperthyroidism, burn, chronic infection, fever, congenital metabolic disorder (cystathionaea, hyperoxaluria, homocystinuria, xanthuremia), congestive heart failure, chronic hemodialysis, malabsorption syndrome with hepatobiliary system disease (such as alcoholism with cirrhosis), intestinal disease (celiac disease, tropical stomatitis enteritis, regional enteritis, persistent diarrhea), and post-gastrectomy. Newborn hereditary vitamin B₆Dependence syndrome.

As a pharmaceutical product, vitamin B is currently used₆The dosage forms on the market comprise tablets and injections. In addition, theVitamin B is added into parenteral nutrition product, health product and feed₆. Vitamin B₆Is unstable to light, heat and strong alkali, so that the product is easy to produce degradation impurities. However, vitamin B is currently recorded in official pharmacopoeia₆The quality standard of the product does not specify specific impurities except EP impurity A and impurity B specified in raw material medicines, and other preparation products do not specify specific impurities. Vitamin B₆The use of injection comprises subcutaneous injection and intravenous injection, which seriously affects the clinical medication safety of the product if the research on impurities is not thorough, and the prior official pharmacopoeias do not strictly limit specific impurities. New dimer of pyridoxol (vitamin B)₆)》 Journal of Organic Chemistry (1969), 34(6), 1993-6、《Analysis of photo- and heat-reaction products of vitamin B₆Bitamin (1999), 73(10), 599-606, JP11209221A and JP11080122A report the degradation route of impurities, but no detailed preparation method of impurities exists, and vitamin B cannot be provided₆Impurity reference substance preparation method necessary for quality control and quality research.

Disclosure of Invention

Aiming at the existing problems, the invention provides a method for separating and preparing the impurity reference substance, and the method is a universal method, is also suitable for separating and preparing other substances which have large polarity and acid condition charge and are difficult to obtain by conventional reverse phase chromatography, normal phase chromatography or directional synthesis, and simultaneously carries out standardization and structure confirmation on the prepared impurity reference substance, namely vitamin B₆The quality research of the bulk drugs and the preparation products provides impurity reference substances, and the quality and the safety of the medicine are improved.

The present invention provides a process for the isolated preparation of compounds having the following characteristics, which compounds tend to have:

(1) large polarity, i.e. LogD distribution coefficient_{(pH1.5-pH7.4)}≤0.0；

(2) Having a dissociation constant of weakly basic or strongly basic;

(3) stable under acidic conditions, and unstable under neutral or alkaline conditions;

(4) acidic conditions generally exist in a charged form;

(5) the conventional reversed phase chromatographic condition under the acidic condition is not properly reserved or not reserved, the peak shape is poor, and accurate quantification is difficult to perform; normal phase chromatography is not suitable because of the large polarity of such materials;

(6) with vitamin B₆For example, the raw material drug is used as a pharmaceutical raw material drug in the form of hydrochloride, and impurities or byproducts generated in the preparation process and the storage process of the raw material drug generally have vitamin B₆Similar or similar structural features and thus similar physicochemical characteristic parameters.

(7) Develop vitamin B with stronger specificity and higher sensitivity₆The method for detecting related substances of the product realizes sensitive and accurate detection of all impurities under the same chromatographic condition.

The invention is as follows in vitamin B₆The preparation method of the substances is detailed by taking the degradation impurities which are most easily generated in the processes of production, storage and use of injection as examples, and the chemical structures of the degradation impurities (formula (II), formula (III), formula (IV) and formula (V)) are as follows:

specifically, the invention is realized by the following technical scheme:

1. vitamin B₆A process for the preparation of an impurity compound comprising the steps of:

1) preparation of vitamin B₆An enriched mother liquor of impurity compounds;

2) detection of vitamin B Using HPLC method₆The formation of impurity compounds;

3) separating the enriched mother liquor obtained in the step 1) by adopting a preparative chromatographic column which takes octadecylsilane chemically bonded silica with polar groups bonded on the surface of the silica as a filler, and simultaneously carrying out qualitative and purity analysis by using a liquid chromatography-mass spectrometer;

4) merging and collecting the high-purity target fractions obtained in the step 3);

5) will step withThe fraction collected in the step 4) is processed under reduced pressure to obtain vitamin B₆A salt-containing product of the impurity compound;

6) desalting the salt-containing product by adopting medium-pressure preparative liquid chromatography, and combining all collected target fractions;

7) concentrating the target fraction obtained in step 6) under reduced pressure to obtain vitamin B₆An impurity compound;

wherein the vitamin B₆The impurity compound is selected from the following compounds:

。

2. vitamin B₆A process for the preparation of an impurity compound comprising the steps of:

1) preparation of vitamin B₆An enriched mother liquor of impurity compounds;

5) carrying out reduced pressure distillation on the fraction collected in the step 4) to obtain vitamin B₆An impurity compound;

wherein the vitamin B₆The impurity compounds are:

3. the method according to

item

1 or 2, wherein the preparing of vitamin B in step 1)₆The enrichment mother liquor of the impurity compounds is carried out under conditions of increased high temperature, light or oxidation.

4. The method of item 3, wherein the elevated temperature is 75 ℃ to 100 ℃.

5. The method of item 4, wherein the elevated temperature is 80 ℃.

6. The method according to

item

1 or 2, wherein the detection conditions for the HPLC method detection in step 2) are as follows: GL Sciences Inertsil ODS-3, 250mm × 4.6mm, 5 μm using octadecylsilane chemically bonded silica as filler; using 0.04% sodium pentane sulfonate solution with pH value adjusted to 2.8 by phosphoric acid as mobile phase A, using methanol as mobile phase B, and carrying out gradient elution; the flow rate was 1.0ml per minute; the detection wavelength is 210 nm; the column temperature is 20 ℃; sample introduction volume is 10 mul

。

7. The method according to

item

1 or 2, wherein the chromatography column in step 3) is a Waters XBridge Prep Shield RP18, 5 μm, 30 x 150mm or a flash column.

8. The method of clause 7, wherein the rapid separation column is a 40g rapid separation column or an 80g rapid separation column.

9. The method according to

item

1 or 2, wherein the step of performing purity analysis by the liquid chromatography-mass spectrometer in the step 3) comprises: taking a proper amount of target fraction collected from a preparative chromatographic column, adding water for dilution to obtain a test solution, taking 10 mu l of the test solution, injecting the test solution into a liquid chromatograph, and taking octadecylsilane chemically bonded silica as a filler; taking 0.01mol/L ammonium formate solution with pH value adjusted to 3.0 by formic acid as a mobile phase A, taking methanol as a mobile phase B, and carrying out gradient elution; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; electrospray positive ion mode (ESI +),

。

10. the method according to

item

1 or 2, wherein the high purity target fraction in the step 4) means a target fraction having a purity of not less than 95%.

11. The process according to item 1, wherein the reduced-pressure treatment in step 5) is concentration under reduced pressure or distillation under reduced pressure.

12. The method according to item 1, wherein the medium pressure preparative liquid chromatography in step 6) is a rapid separation column using octadecylsilane-bonded silica gel of 20 to 35 μm as a packing or Waters Xbridge Prep Shield RP18, 5 μm, 30X 150mm, and water and methanol or acetonitrile are used as mobile phases, and the flow rate is set to 20 to 25 ml/min.

13. The method of claim 12, wherein the mobile phase is water and methanol, and the flow rate is set to 20 ml/min.

14. The method according to item 6, wherein the mobile phase aqueous phase adopted by the chromatographic column in the step 3) is an aqueous solution of a buffer salt or an ion-pair reagent.

15. The method of claim 14, wherein the buffer salt comprises formic acid or formate, trifluoroacetic acid, acetic acid or acetate, phosphoric acid or phosphate, citric acid or citrate, and the salt comprises potassium, sodium, ammonium salts.

16. The method of claim 14, wherein the ion pairing agent is an alkyl sulfonate.

17. The method of item 16, wherein the alkyl sulfonate is heptane sulfonate, pentane sulfonate, or octane sulfonate.

18. The method according to item 14, wherein the chromatography column in step 3) employs a flow rate of 10 to 30ml/min, preferably 15 to 25ml/min, more preferably 15 to 20 ml/min.

19. The method according to claim 18, wherein the mobile phase aqueous phase used in the chromatography column of step 3) is an ammonium formate solution.

20. The method according to item 17, wherein the mobile phase aqueous phase adopted by the chromatographic column in the step 3) is a sodium pentanesulfonate solution.

21. The method of item 19, wherein the ammonium formate is at a concentration of 1mM to 500mM, preferably 10mM to 50mM, more preferably 10 mM.

22. The method according to item 6, wherein the column of step 3) is Waters Xbridge Prep Shield RP18, 5 μm, 30X 150mM, and the mobile phase is 10mM formazanAmmonium salt solution of said vitamin B₆The impurity compounds are:

。

23. the method according to item 6, wherein the chromatographic column in step 3) is a rapid separation column, the mobile phase A is 0.04% sodium pentanesulfonate solution, the phase B is methanol, gradient elution is carried out, the detection wavelengths are 210nm and 290nm, and the flow rate is 20ml/min, wherein the vitamin B₆The impurity compounds are:

。

24. a compound of formula II in vitamin B₆The application of the product as an impurity reference substance in impurity inspection,

formula (II).

25. A compound of formula III in vitamin B₆The application of the product as an impurity reference substance in impurity inspection,

formula (III).

26. A compound of formula IV in vitamin B₆The application of the product as an impurity reference substance in impurity inspection,

formula (IV).

27. A compound of formula V in vitamin B₆The application of the product as an impurity reference substance in impurity inspection,

formula (V).

28. The use according to any one of claims 24-27, wherein the vitamin B is₆The HPLC conditions for detecting the substances are as follows: chromatographic column using octadecylsilane chemically bonded silica as filler; using 0.04% sodium pentane sulfonate solution with pH value adjusted to 2.8 by phosphoric acid as mobile phase A, using methanol as mobile phase B, and carrying out gradient elution; the flow rate was 1.0ml per minute; the detection wavelength is 210 nm; the column temperature is 20 ℃; sample introduction volume is 10 mul

。

29. The use according to item 28, wherein the chromatography column is GL Sciences Inertsil ODS-3, 250mm x 4.6mm, 5 μm.

30. Vitamin B₆A detection method of related substances, wherein the detection method adopts high performance liquid chromatography to detect vitamin B₆And impurities in the preparation are detected, and the high performance liquid chromatography conditions comprise: chromatographic column using octadecylsilane chemically bonded silica as filler; using 0.04% sodium pentane sulfonate solution with pH value adjusted to 2.8 by phosphoric acid as mobile phase A, using methanol as mobile phase B, and carrying out gradient elution; the flow rate was 1.0ml per minute; the detection wavelength is 210 nm; the column temperature is 20 ℃; sample introduction volume is 10 mul

。

31. The method of item 30, wherein the chromatography column is GL Sciences insetsil ODS-3, 250mm x 4.6mm, 5 μ ι η.

Compared with the prior art, the invention has the following beneficial technical effects:

the impurity referred to in Table 1 is vitamin B₆The impurities which are easily generated in the process of storing the injection are not strictly limited according to the specific impurities in the official pharmacopoeias, and the impurities are not sold in the current market. To ensure the medicineThe safety of the product in the using process and detailed and thorough impurity research are very necessary. The information of related literatures is consulted, only the degradation route of impurities is reported, and the preparation method is not detailed. Aiming at impurities in table 1, the invention provides vitamin B₆The preparation, separation and purification method of the reference substance for degrading impurities can prepare high-purity B rapidly and massively at low cost₆Degrading impurity reference substance, solving the problem of vitamin B₆The quality control of raw materials and preparations thereof and the lack of impurity reference substances in the research of impurities. For further improving vitamin B₆And the quality safety of the preparation product, the physical health of the consumer and the like, and has important practical significance.

Drawings

FIG. 1 is a HPLC chart of an enriched mother liquor of a compound of formula (II).

FIG. 2 is a HPLC plot of an enriched mother liquor of a compound of formula (III).

FIG. 3 is a HPLC chart of the enriched mother liquor of the compound of formula (IV).

FIG. 4 is a HPLC chart of an enriched mother liquor of the compound of formula (V).

FIG. 5 shows a typical spectrum of the preparative separation of the compound of formula (II).

FIG. 6 shows a typical spectrum of the preparation and separation of the compound of formula (III).

FIG. 7 shows typical spectra for preparative separation of compounds of formula (IV) and formula (V).

Fig. 8 is a mass spectrum (positive and negative ion mode) of the compound of formula (II), wherein fig. 8A is the positive ion mode and fig. 8B is the negative ion mode.

FIG. 9 shows the NMR 1HNMR spectrum of the compound of formula (II).

FIG. 10 shows NMR 13C NMR spectra of compounds of formula (II).

Fig. 11 is a mass spectrum (positive and negative ion mode) of the compound of formula (III), wherein fig. 11A is the positive ion mode and fig. 11B is the negative ion mode.

FIG. 12 shows the NMR 1HNMR spectrum of the compound of formula (III).

FIG. 13 is a NMR 13CNMR spectrum of the compound of formula (III).

FIG. 14 is a nuclear magnetic resonance two-dimensional nuclear magnetic HMBC spectrum of the compound of formula (III).

Fig. 15 is a mass spectrum (positive and negative ion mode) of the compound of formula (IV), wherein fig. 15A is the positive ion mode and fig. 15B is the negative ion mode.

FIG. 16 shows the NMR 1HNMR spectrum of the compound of formula (IV).

FIG. 17 shows NMR 13C NMR spectra of compounds of formula (IV).

FIG. 18 is a nuclear magnetic resonance two-dimensional nuclear magnetic HMBC spectrum of the compound of formula (IV).

FIG. 19 shows the NMR two-dimensional NMR HSQC spectrum of the compound of formula (IV).

Fig. 20 is a mass spectrum (positive and negative ion mode) of the compound of formula (V), wherein fig. 20A is the positive ion mode and fig. 20B is the negative ion mode.

FIG. 21 shows the NMR 1HNMR spectrum of the compound of formula (V).

FIG. 22 shows NMR 13C NMR spectra of compounds of formula (V).

FIG. 23 is a nuclear magnetic resonance two-dimensional nuclear magnetic HMBC spectrum of the compound of formula (V).

FIG. 24 shows the NMR two-dimensional NMR HSQC spectrum of the compound of formula (V).

FIG. 25 shows vitamin B prepared by a user₆The related substance map of the injection is 40 ℃/RH75 percent for three months.

FIG. 26 shows vitamin B₆The related substance map of the injection reference preparation is 40 ℃/RH75 percent for three months.

FIG. 27 shows vitamin B prepared by a user₆The injection is exposed to light for 5 days to obtain the related substance map.

FIG. 28 is a map of the suitability of the system for detecting a substance of interest using the assay of the invention.

FIG. 29 is a spectrum of a substance of interest in a reference preparation for detecting the substance of interest using the assay of the invention.

FIG. 30 is a chart of systematic applicability of the method for detecting related substances using the Chinese pharmacopoeia analysis method.

FIG. 31 is a reference preparation related substance profile for the detection of related substances using the Chinese pharmacopoeia analysis method.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Vitamin B for use in the present invention₆Bulk drug, self-made vitamin B₆Injection and vitamin B₆The reference preparations are all donated by Chinese tsukamur pharmacy. Other raw materials, reagents and solvents used in the present invention are not particularly limited, and commercially available conventional raw materials, reagents and solvents can be used. The columns or packing materials were purchased from Bonajier, Waters, Agilent, Inc. or their agents.

Separation equipment used in impurity preparation:

high pressure preparative High Performance Liquid Chromatography (HPLC): a Waters automated purification system comprising a 2489UV/Visible detector, a 2545 binary high pressure gradient pump, a 2767 automated sample collector, and a Masslyn4.0 workstation. Or an instrument device functionally equivalent thereto.

Medium pressure preparative liquid chromatography: the CHEETAH automatic purification system (MP 200) produced by Bora and Aijier consists of an ultraviolet detector, a binary medium pressure pump, a manual sample introduction system and a collection platform. Or instrumentation functionally equivalent thereto.

The instrument and method for data acquisition:

mass spectrum collection adopts Agilent Infinity Series 1290-6120Quadrupole LC-MS, ESI source and positive/negative ion mode; the detailed configuration is as follows: G4225A 1260 online degasser, G1312B 1260 binary gradient pump, G1367E1260 high pressure autosampler, G1330B 1290 column oven and G1316C 1290 detector, the data acquisition workstation is:

nuclear magnetic resonance hydrogen spectroscopy (HNMR) data was acquired using the instrument models: BrukerAVANCE600, resonance frequency: 600MHz, using solvent: the compounds of formula (II) to formula (IV) are D₂O; the compound of formula (V) is deuterated dimethyl sulfoxide.

Nuclear magnetic resonance was entrusted to Tianjin Han Union testing technology (Tianjin) Limited.

Formula (II) - (V) is vitamin B₆The raw material medicine or preparation product is slowly generated under the conditions of high temperature, illumination or oxidation, so that in order to obtain a sample containing high content of the formula (II) -formula (V), the sample with higher content of the formula (II) -formula (V) is obtained by intensifying the conditions of high temperature, illumination or oxidation according to different degradation ways of the formula (II) -formula (V), and the reference product of the formula (II) -formula (V) is further obtained by separation and purification.

EXAMPLE 1 preparation and characterization of Compounds of formula (II)

First, preparation of enriched mother liquor

Preparing an enriched mother liquor containing a compound of formula (II) above: collecting vitamin B₆About 2g of raw material medicine, 30% H₂O₂Heating in 80 deg.C water bath for 8 hr to give 10ml solution, taking out, oven standing at 60 deg.C overnight, and adding 30% H₂O₂Repeating 3 cycles from about 10 ml' to obtain the enriched mother liquor of the compound of the formula (II), wherein the enriched mother liquor is yellow clear liquid and is stored in a sealed manner at the temperature of 2-8 ℃.

Alternatively, the bath temperature may be varied within a suitable range, such as 75 ℃ to 100 ℃, H₂O₂Can also be varied within suitable ranges, such as 8% -30% H₂O₂。

HPLC analysis method of enriched mother liquor of formula (II)

After taking the enriched mother liquor of the compound of formula (II), diluting it with water to an appropriate concentration, detecting the formation of the compound of formula (II) by HPLC method and estimating its content by area normalization, the HPLC chromatogram is shown in FIG. 1. The content of the compound shown in the formula (II) is about 28-34% by adopting an area normalization method. Liquid chromatography relative (VB)₆) The retention time was 0.39.

Chromatographic conditions are as follows: octadecylsilane bonded silica gel as filler (GL Sciences Inertsil ODS-3, 250 mm. times.4.6 mm, 5 μm); gradient elution was performed according to table 2 with 0.04% pentane sodium sulfonate solution (pH adjusted to 2.8 with phosphoric acid) as mobile phase a and methanol as mobile phase B; the flow rate was 1.0ml per minute; the detection wavelength is 210 nm; the column temperature is 20 ℃; the injection volume was 10. mu.l.

。

Under the chromatographic conditions, VB₆The retention time of (a) is about 22 minutes. The chromatographic conditions are ion pair based reverse phase HPLC analysis methods, which are very challenging for preparative separation of impurities.

Thirdly, separation and purification of the compound of the formula (II)

1) Initial separation, confirmation and post-treatment of enriched mother liquor

As can be seen from FIG. 1, it is rich in vitamin B as the main component in the mother liquor₆Essentially all was destroyed, the normalized content of formula (II) in the enriched mother liquor was higher, about 34%, with a retention time of about 8.7 minutes. According to the analysis chromatographic condition of the crude mother liquor and the retention time of the formula (II) under the condition, the separation preparation is carried out by using a Waters high-pressure preparation liquid phase.

The chromatographic column adopts a preparative chromatographic column using octadecylsilane chemically bonded silica with polar groups bonded on the surface of the silica as a filler, such as Waters Xbridge Prep Shield RP18, specification: 5 μm, 30X 150mm, which has better retention of large polar substances and good peak shape.

Chromatographic conditions for preparative separation: to obtain a good chromatographic peak profile, the mobile phase aqueous phase is preferably a buffered salt system, and a 10mM ammonium formate solution was selected as the mobile phase in this experiment. The flow rate was 15 ml/min. The detection wavelength is 210nm and 220 nm. The mother liquor of the compound shown in the formula (II) is not concentrated, is directly injected and separated, the injection volume is 4ml, and pure water is adopted for isocratic elution for 16 minutes. The threshold value for starting collection is set, the collection is automatic, and all components with the content larger than the collection threshold value in the mother liquor of the formula (II) are automatically collected in different test tubes by an instrument. The peak time of the compound of formula (II) under the preparation conditions was about 10 min. A typical separation profile is shown in fig. 5. Subjecting the target fraction of the compound of formula (II), i.e. the receiving time, to a test of about 9.2min to 10.2minPerforming purity and qualitative analysis on the sample in the tube by using a liquid chromatography-mass spectrometer, wherein the main peak of the target fraction is required to be in a positive ion modem/zAbout 158%, and the purity of the main peak is not lower than 95%, and according to the detection result, the collection threshold can be properly adjusted to make the target fraction meet the requirements.

The analysis conditions of the liquid chromatography-mass spectrometry are as follows:

collecting appropriate amount (about 3-5 drops) of the collected target fraction, and diluting with water to obtain sample solution if the target peak in the separation spectrogram is a flat head peak. Performing high performance liquid chromatography-mass spectrometry (China pharmacopoeia 2015 edition fourth part general rules 0512 and 0431) test, and using octadecylsilane chemically bonded silica as filler; gradient elution is carried out according to the table 3 by taking 0.01mol/L ammonium formate solution (pH value is adjusted to 3.0 by formic acid) as a mobile phase A and methanol as a mobile phase B; the flow rate was 1.0ml per minute; the column temperature is 30 ℃; a mass spectrum detector is adopted, an electrospray positive ion mode (ESI +) is adopted, 10 mu l of test solution is taken and injected into the mass spectrum detector, and a mass spectrum is recorded, wherein the mass-to-charge ratio (m/z) of a main peak is about 158.0.

。

All the enriched mother liquors of formula (II) were put in Waters high pressure to prepare liquid phase, and automatic sample separation was performed, with an autoinjection volume of 2ml and automatic collection of 480 test tubes (15 mm. 150 mm).

All target fraction samples with the purity of formula (II) being more than 95% are combined, and concentrated to 10-20 ml at 35-40 ℃ under reduced pressure (oil pump: -0.095-0.10 mpa), so as to obtain the salt-containing product of formula (II).

2) Desalting of salt-containing product of formula (II)

Ammonium formate is used in the mobile phase during the initial enrichment of the mother liquor of formula (II), and therefore the salt-containing product of formula (II) contains a large amount of ammonium formate. Preparing liquid phase by adopting medium pressure, and desalting. The chromatographic conditions are as follows:

and (2) taking water and methanol or acetonitrile as mobile phases, taking a 40g rapid separation column with octadecylsilane chemically bonded silica as a filler (20-35 mu m) as a chromatographic column, setting the detection wavelengths to be 210nm and 254nm, and setting the flow rate to be 20 ml/min. And (3) carrying out isocratic elution for 3-5 minutes by using a high-proportion water phase, such as a 98% water phase. The organic phase is preferably selected to be methanol, due to the greater polarity of formula (II). And then increasing the proportion of the organic phase (methanol) to 40% within 5-10 min, collecting a target peak, and directly injecting the salt-containing product of the formula (II) with the sample loading volume of 2-3 ml. And after each sample injection collection is finished, quickly returning to the initial organic phase proportion to perform chromatographic column balance for 7-10 minutes, and desalting all the salt-containing product solution of the formula (II) by using the method. All collected target fractions are combined, and concentrated at 35-40 ℃ under reduced pressure (oil pump: -0.095-0.10 mpa) to obtain a light yellow oily substance which is the compound of the formula (II).

Alternatively, an 80g flash column with octadecylsilane chemically bonded silica as packing (20-35 μm) can be selected as the chromatographic column, the flow rate can be changed within a proper range, such as 20-25 ml/min, and the sample loading volume can be changed within a proper range, such as 2-3-4-6 ml.

In addition, in order to obtain higher purification effect, high pressure preparative liquid chromatography such as Waters Xbridge Prep Shield RP18, 5 μm, 30X 150mm can also be used.

Four, standardization of Compound of formula (II)

The purity of the formula (II) is standardized by liquid chromatography in parallel by preparing 6 samples for double persons and different instruments, and taking the average value of 12 samples as the final purity.

The retention time of the compound of formula (II) under the chromatography conditions of the compound of formula (II) enriched mother liquor is about 8.2 minutes, so the equilibrium time is 10 minutes except for the equilibration process where the gradient elution procedure is returned to the initial organic phase ratio after 25 minutes. After calibration, the liquid chromatography purity of the control of formula (II) was 96.23% (210 nm).

Fifthly, structural identification of the compound of the formula (II)

And (3) structural identification: in addition to confirmation of the retention time of the chromatogram, a mass spectrum, a nuclear magnetic resonance hydrogen spectrum, and a carbon spectrum were also performed.

The spectrum of formula (II) shows the molecular ion peakm/z = 157.9 [M+H]⁺/155.9 [M-H]^-The corresponding molecular weight corresponds to the theoretical molecular weight 157.12 of the molecular formula provided (C)₆H₇NO₄) The spectra are shown in FIG. 8. The nuclear magnetic resonance hydrogen spectrum and carbon spectrum of the compound of formula (II) are shown in figures 9-10.

EXAMPLE 2 preparation and characterization of Compounds of formula (III)

First, preparation of enriched mother liquor

Collecting vitamin B₆Dissolving 1g of raw material medicine in 500ml of water, adjusting the pH value to 5.0-6.0 by using 1mol/L of sodium hydroxide, charging nitrogen for 30-60 min, sealing, and illuminating for 20-30 days to obtain an enriched mother solution of the compound shown in the formula (III), wherein the enriched mother solution is a yellow clear liquid and is stored at room temperature. To enrich enough crude product, 10 mother liquors were prepared in parallel.

HPLC analysis method of enriched mother liquor of formula (III)

And (3) directly injecting and analyzing the enriched mother liquor of the compound shown in the formula (III) by adopting an HPLC method, detecting the generation condition of the compound shown in the formula (III) and estimating the content of the compound by using an area normalization method, wherein an HPLC spectrogram is shown in figure 2. The compound of the formula (III) is gradually increased along with the prolonging of the illumination time, the illumination is carried out for 20-30 days, and the generation amount is 23% -36% measured by an HPLC area normalization method. Liquid chromatography relative (VB)₆) The retention time was 0.87. The specific experimental conditions and methods were the same as those of HPLC analysis of the mother liquor of the compound of formula (II) in example 1.

Thirdly, separation and purification of the compound of the formula (III)

After the enriched mother liquor of the formula (III) is properly concentrated, a CHEETAH automatic purification system (MP 200) produced by Bora-Ejeger is adopted to carry out primary separation on the enriched mother liquor of the formula (III). A40 g or 80g fast separation column using octadecylsilane chemically bonded silica as a filler (20-35 μm) is used as a stationary phase, and a gradient elution procedure can be finely adjusted. 10mM ammonium formate solution was selected as the mobile phase for this experiment (pH 4.8 adjusted with formic acid or ammonia). Methanol was used as the organic phase. The flow rate is 20ml/min-25ml/min, the detection wavelength is 210nm and 260nm, the mixture is eluted with 88% water phase for 17 minutes at equal intervals, the organic phase ratio is increased from 12% to 70% after a target peak is collected (14 minutes-18 minutes), the organic phase ratio is decreased to 12% after the target peak is maintained for 3 minutes, the organic phase ratio is balanced for 7-10 minutes, and a separation procedure is completed. A typical separation profile is shown in figure 6.

Alternatively, an 80g flash column packed with octadecylsilane bonded silica (20-35 μm) can be selected as the chromatographic column, and the flow rate can be varied within a suitable range, such as 20-25 ml/min.

Purity and mass spectrum confirmation of the target fraction: taking appropriate amount (about 3-5 drops) of the collected liquid, if the target peak in the separation spectrogram is a flat head peak, adding water for appropriate dilution, and shaking up to obtain the sample solution. When analyzed according to the conditions identified in the mass spectrum of formula (II), the mass-to-charge ratio (m/z) of the main peak should be about 170.1, and the purity of the main peak should be not less than 95%. And repeatedly injecting the residual enrichment mother liquor according to the separation procedure.

And (3) adding dilute ammonia water or dilute formic acid into the target fraction with the purity of more than 95% to adjust the pH value to 5.5-6.0, carrying out reduced pressure distillation and concentration (40 ℃ and-0.08 Mpa), and removing the solvent to obtain a light yellow solid, namely the salt-containing product of the compound shown in the formula (III).

2) Desalting of salt-containing product of Compound of formula (III)

The salt-containing product of the compound of formula (III) contains ammonium formate and is desalted using the CHEETAH automatic purification system (MP 200) from bornaegel. The chromatographic conditions are as follows: and (2) taking water and methanol or acetonitrile as mobile phases, taking a 40g rapid separation column with octadecylsilane chemically bonded silica as a filler (20-35 mu m) as a chromatographic column, setting the detection wavelengths to be 210nm and 260nm, and setting the flow rate to be 20 ml/min. The solid is dissolved into a solution with the concentration of about 30mg/ml by adding water, the initial organic phase proportion is 10 percent, the linear rise is carried out to 90 percent in 15 minutes, and the target fraction is collected, and the injection volume is 2 ml. After the target fraction peak is collected, the proportion of the organic phase is adjusted to 10%, and the organic phase is balanced for 7-10 minutes, so that a desalting procedure is completed. The remaining samples were all desalted following this procedure. And (3) combining all target fractions, adding hydrochloric acid to adjust the pH value to 5.5-6.0, and concentrating under reduced pressure (40 ℃ and-0.08 Mpa) to remove all solvents, or concentrating under reduced pressure to remove most organic solvents and freeze-drying to obtain a white-like solid.

Alternatively, an 80g flash column with octadecylsilane chemically bonded silica as packing (20-35 μm) can be selected as the chromatographic column, the flow rate can be varied within a suitable range, such as 20-25 ml/min, and the sample loading volume can be varied within a suitable range, such as 2-5 ml.

Fourth, standardization of Compound of formula (III)

The liquid chromatography is adopted for double persons, different instruments and 6 samples are respectively prepared in parallel for purity calibration, and the average value of 12 samples is taken as the final purity.

The retention time of the compound of formula (III) under the chromatography conditions of the compound of formula (III) enriched mother liquor is about 16.8 minutes, so the equilibrium time is 10 minutes, except for the equilibration process where the gradient elution procedure is returned to the initial organic phase ratio after 25 minutes. After standardization, the liquid chromatography purity of the control of formula (III) was 99.63% (210 nm).

Fifthly, structural identification of the compound of the formula (III)

And (3) structural identification: in addition to confirmation of the retention time of the chromatogram, a mass spectrum, a nuclear magnetic resonance hydrogen spectrum, a carbon spectrum, and a two-dimensional nuclear magnetic spectrum were also performed.

The spectrum of formula (III) shows molecular ion peaks M/z = 169.9 [ M + H ] +/168.0 [ M-H ] -, corresponding to molecular weights consistent with the theoretical molecular weight of formula 169.18 (C8H 11NO 3) provided in figure 11. The hydrogen and carbon nuclear magnetic resonance spectrum and the two-dimensional nuclear magnetic HMBC spectrum of the formula (III) are shown in attached figures 12-14. The spectrogram analysis is as follows:

the nuclear magnetic resonance hydrogen spectrum and carbon spectrum data and the attribution are as follows:

two-dimensional nuclear magnetic HMBC spectra, showing the following remotely related test data for hydrocarbons:

EXAMPLE 3 preparation and characterization of Compounds of formula (IV)

First, preparation of enriched mother liquor

Collecting vitamin B₆Dissolving 2g of raw material medicine in about 40ml of water, and adjusting the pH value to 3.0-5.0 by using 1mol/L of sodium hydroxide. And (3) placing the solution in a penicillin bottle, sealing by rolling a cover, and sterilizing at 121 ℃ for 1-2 hours to obtain the enriched mother liquor of the compound shown in the formula (IV), wherein the enriched mother liquor is a light yellow clear liquid and is stored at room temperature.

HPLC analysis method of compound mother liquor of formula (IV)

After taking the enriched mother liquor of the compound of formula (IV) and diluting it with water to an appropriate concentration, the formation of formula (IV) is detected by HPLC method and its content is estimated by area normalization, and the HPLC chromatogram is shown in FIG. 3. The compound of formula (IV) is present in a sterilized solution for 2 hours in an amount of about 6% to about 7% by liquid chromatography Versus (VB)₆) The retention time was 1.39. The experimental conditions and methods were the same as those of HPLC analysis of the mother liquor of formula (II) in example 1.

Thirdly, separation and purification of the compound of the formula (IV)

The enriched mother liquor of formula (IV) is subjected to preliminary separation by adopting a CHEETAH automatic purification system (MP 200) produced by Bora-Aijier. Taking 40g of a rapid separation column using octadecylsilane chemically bonded silica as a filler (20-35 mu m) as a stationary phase, taking a mobile phase A as a 0.04% sodium pentanesulfonate solution (pH2.8), taking a phase B as methanol, carrying out gradient elution according to the following table, wherein the detection wavelengths are 210nm and 290nm, and the flow rate is 20 ml/min.

The separation gradient elution procedure was as follows:

。

alternatively, an 80g flash column packed with octadecylsilane chemically bonded silica (20-35 μm) may be selected as the chromatographic column, the flow rate may be varied within a suitable range, such as 15ml/min-20ml/min, and the pH of the sodium pentanesulfonate solution may be varied within a suitable range, such as pH 2.5-4.0.

And (3) taking 1-3 ml of the enriched mother liquor of the compound shown in the formula (IV), loading, collecting fractions for 30-36 min, and carrying out qualitative confirmation by using a liquid chromatography-mass spectrometer. The analytical method of the LC-MS is the same as the chromatographic condition under the formula (II). According to the analysis result of the liquid chromatograph-mass spectrometer, the mass-to-charge ratio (M/z) of the main peak of the fraction of the formula (IV) is 320.9 [ M + H ]]⁺/ 318.9 [M-H]The purity of the main peak is not lower than 90%. A typical separation profile is shown in fig. 7.

The above separation procedure was repeated, and the fractions of formula (IV) were collected and combined, adjusted to pH about 3.5-4.5 with dilute ammonia water, and distilled at room temperature under reduced pressure (-0.08 MPa) to remove all the solvent to give a pale yellow solid.

The chemical structure of formula (IV), which is inferred from the mass spectrometric information, is easily salified with sodium pentanesulfonate, and therefore, no desalting treatment is performed.

The purity of the compound of formula (IV) was confirmed by the same method as the enriched solution analysis. The purity of formula (IV) was 95.12%.

Structural identification of compound of formula (IV)

And (3) structural identification: in addition to confirmation of the retention time of the chromatogram, mass spectra, nuclear magnetic resonance hydrogen spectra, carbon spectra, two-dimensional nuclear magnetic HSQC spectra, and HMBC spectra were also performed.

The spectrum of formula (IV) shows the molecular ion peakm/z = 320.9 [M+H]⁺/ 318.9 [M-H]^-The corresponding molecular weight is 320.34 (C) which corresponds to the theoretical molecular weight of the base of the compound supplied (C)₁₆H₂₀N₂O₅) The map is shown in figure 15. The nuclear magnetic resonance hydrogen and carbon spectrum, two-dimensional nuclear magnetic HSQC spectrum and HMBC spectrum of the compound shown in the formula (IV) are shown in the attached figures 16-19.

two-dimensional nuclear magnetic HSQC spectrum and HMBC spectrum, and the shown hydrocarbon correlation relationship is as follows:

EXAMPLE 4 preparation and characterization of Compounds of formula (V)

First, preparation of enriched mother liquor

Collecting vitamin B₆Dissolving 2g of raw material medicine in about 40ml of water, and adjusting the pH value to 3.0-5.0 by using 1mol/L of sodium hydroxide. And (3) placing the solution in a penicillin bottle, sealing, sterilizing at 121 ℃ for 6 hours to obtain an enriched mother solution of the compound shown in the formula (V), wherein the enriched mother solution is a light yellow clear liquid, and storing at room temperature.

Second, HPLC analysis method of mother liquor of formula (V)

After taking the enriched mother liquor of the compound of formula (V), diluting it with water to an appropriate concentration, the formation of formula (V) is detected by HPLC method and its content is estimated by area normalization, and the HPLC chromatogram is shown in FIG. 4. The compound of formula (V) has an HPLC normalized content of about 14% in a solution sterilized for 6 hours, relative to liquid chromatography (VB)₆) The retention time was 2.11. The experimental conditions and methods were the same as those of HPLC analysis of the mother liquor of formula (II) in example 1.

Thirdly, separation and purification of the compound of the formula (V)

The enriched mother liquor of formula (V) was subjected to preliminary separation using the CHEETAH automated purification system (MP 200) produced by bornaegel. Taking 40g of a rapid separation column which takes octadecylsilane chemically bonded silica as a filler (20-35 mu m) as a stationary phase, taking a mobile phase A as a 0.04% sodium pentanesulfonate solution (pH2.5-3.5), taking a phase B as methanol, carrying out gradient elution according to the following table, wherein the detection wavelengths are 210nm and 290nm, and the flow rate is 20ml/min-25 ml/min.

The separation gradient elution procedure was as follows:

。

and (3) taking 1ml of the enrichment mother liquor of the compound shown in the formula (V), loading, collecting fractions of 44-49 min, and carrying out qualitative confirmation by using a liquid chromatography-mass spectrometer. The analytical method of the LC-MS is the same as the chromatographic conditions under the formula (II). According to the analysis result of the liquid chromatograph-mass spectrometer, the mass-to-charge ratio (m/z) of the main peak of the fraction shown in the formula (V) is 321.1, and the purity of the main peak is not lower than 95%.

The above separation procedure was repeated, fractions of the compound of formula (V) having a purity of more than 95% were combined, adjusted to about pH7.0 with 0.1M sodium hydroxide solution, and distilled under reduced pressure (40 ℃ C., -0.08 MPa) to remove all the solvent to give a pale yellow solid as a salt-containing product of the compound of formula (V).

2) Desalting of salt-containing product of Compound of formula (V)

The salt-containing product of the compound shown in the formula (V) contains inorganic salts such as sodium pentanesulfonate, sodium hydroxide and the like, and the CHEETAH automatic purification system (MP 200) produced by BornaeIjer is adopted for desalting. The chromatographic conditions are as follows: and (2) taking water and methanol or acetonitrile as mobile phases, taking a 40g rapid separation column with octadecylsilane chemically bonded silica as a filler (20-35 mu m) as a chromatographic column, setting the detection wavelengths to be 210nm and 260nm, and setting the flow rate to be 20 ml/min. The solid was dissolved in methanol-water (1: 1) to a concentration of about 50mg/ml, the initial organic phase ratio was 10%, the linear rise was 10 minutes to 70%, and the target fraction was collected in a sample volume of 2 ml. After the target fraction peak is collected, the proportion of the organic phase is adjusted to 10%, and the organic phase is balanced for 6-10 minutes, so that a desalting procedure is completed. The remaining samples were all desalted following this procedure. All the target fractions were combined and concentrated under reduced pressure (40 ℃ C., -0.08 MPa) to remove all the solvent, or concentrated under reduced pressure to remove most of the organic solvent and then lyophilized to give a pale yellow solid.

The purity of the compound of formula (V) was confirmed by the same method as the enriched solution analysis. The purity of formula (V) is about 95.78%.

Alternatively, an 80g flash column packed with octadecylsilane chemically bonded silica (20-35 μm) can be selected as the chromatographic column, the flow rate can be varied within a suitable range, such as 20-25 ml/min, and the loading volume can be varied within a suitable range, such as 2-5 ml.

Standardization of Compound of formula (V)

The liquid chromatography is adopted for double persons, different instruments and 6 samples are respectively prepared in parallel for purity calibration, and the average value of 12 samples is taken as the final purity. The chromatographic condition is confirmed by the same method as the enriched liquid analysis. After standardization, the control of formula (V) had a purity of about 95.78%.

Fifthly, structural identification of the compound of the formula (V)

The spectrum of formula (V) shows molecular ion peak M/z = 320.9 [ M + H ]]⁺/ 318.9 [M-H]^-The corresponding molecular weight corresponds to the theoretical molecular weight 320.34 of the molecular formula provided (C)₁₆H₂₀N₂O₅) The spectra are shown in FIG. 20. The hydrogen and carbon spectra, two-dimensional nuclear magnetic HSQC spectra and HMBC spectra of the nuclear magnetic resonance shown in the formula (V) are shown in attached figures 21-24.

example 5 application of Compounds of formula (II) to formula (V) as impurity reference

Performing high performance liquid chromatography (China pharmacopoeia 2020 edition four ministry of general rules 0512).

Test solution: collecting vitamin B₆The injection solution is prepared into a solution containing about 2mg per 1ml by diluting with water, wherein the solution is used as each test sample solution, and the solution comprises a 0-month sample of the injection solution, a 3-month sample of accelerated stability research (accelerated test condition: 40 ℃/RH 75%), a 3-month sample of accelerated stability research reference preparation (accelerated test condition: 40 ℃/RH 75%), and a proper amount of influencing factor research illumination (test condition: illumination intensity 5000 LX). An appropriate amount of each sample solution was precisely measured and diluted with water to give a solution containing about 2. mu.g of each 1ml as a control solution.

System applicability solution: respectively dissolving appropriate amount of externally purchased known impurities ZZ-01, ZZ-02, ZZ-12, pyridoxal, RRT0.39 (formula II), RRT0.87 (formula III) and RRT2.11 (formula V) in water (ZZ-01 is dissolved by adding appropriate amount of DMSO if necessary, and ZZ-02 and RRT2.11 are dissolved by adding appropriate amount of methanol) to obtain solution containing about 1mg per 1ml, and storing as component control stock solution. Collecting vitamin B₆The reference substance and appropriate amount of reference substance stock solution of each component are dissolved in water and diluted to obtain a solution containing vitamin B per 1ml₆2mg, about 2. mu.g of each impurityThe mixed solution of (1).

Chromatographic conditions and an impurity enriched liquid analysis method.

An analytical instrument, namely a Waters high performance liquid chromatograph or an Agilent high performance liquid chromatograph, is provided with an ultraviolet or DAD detector, a column incubator, an automatic sample injector, a high-pressure binary pump or a low-pressure quaternary pump system.

And (3) taking 10 mu l of the system applicability solution, injecting the solution into a liquid chromatograph, and recording a spectrogram. The peak output sequence is as follows: RRT0.39, pyridoxal, RRT0.87, ZZ-12, vitamin B₆ZZ-01, ZZ-02 and RRT2.11, the separation degree between pyridoxal peak and RRT0.87 peak is not less than 1.0, the separation degree between other component peaks is not less than 1.5, and the theoretical plate number is according to vitamin B₆The peak calculation should be no less than 5000.

Precisely measuring each control solution and sample solution by 10 μ l, injecting into a liquid chromatograph, recording chromatogram, and calculating the content of each impurity according to the main component self-control method, wherein the result is shown in the following table. HPLC spectrograms of the home-made and reference preparations accelerated for 3 months and the home-made preparations were exposed to light for 5 days are shown in the attached figures 25-27.

As can be seen from the above experimental results, vitamin B was accelerated under the test conditions₆The injection mainly degrades impurities RRT1.39 (formula IV) and RRT2.11 (formula V), and degrades impurities RRT0.39 (formula II) and RRT0.87 (formula III) under illumination condition, and vitamin B₆In the process of production and storage, the injection is very easily affected by temperature and illumination, so that the impurities are generated, and in order to effectively control the product quality and ensure the medication safety, the research of mainly degrading the impurities RRT0.39 (formula II), RRT0.87 (formula III), RRT1.39 (formula IV) and RRT2.11 (formula V) is very necessary.

Example 6: vitamin B₆Comparison of detection methods of related substances in injection

The vitamin B6 injection is loaded in the second part of the Chinese pharmacopoeia 2020, European pharmacopoeia EP10.0, wherein vitamin B is also specified₆Detection of injection-related substancesMethod, however, the method for detecting related substances in vitamin B6 injection loaded in EP10.0 adopts octadecylsilane chemically bonded silica as a filler (250 mm × 4.6mm, 5 μm) as a stationary phase, and 100% potassium dihydrogen phosphate solution (2.72 g of potassium dihydrogen phosphate is taken, 900ml of water is added for dissolution, the pH is adjusted to 3.0 by phosphoric acid, and water is added for dilution to 1000 ml) with pH of 3.0 as a mobile phase for isocratic elution, wherein the detection wavelength is 210 nm;

china pharmacopoeia 2020 edition two vitamin B₆The detection method of related substances of the injection adopts octadecylsilane chemically bonded silica as a filler (250 mm multiplied by 4.6mm, 5 mu m) as a stationary phase, 0.04% sodium pentane sulfonate solution (pH value is adjusted to 3.0 by glacial acetic acid) as a mobile phase A, methanol as a phase B, and the ratio of A: b, 85: 15 (V: V) was subjected to isocratic elution. The detection wavelength is 291 nm;

since the analysis method of EP10.0 is a pure water phase, no organic phase is added, and many degradation impurities cannot be effectively separated and eluted under the method, the detection results are compared with the Chinese pharmacopoeia method in an important point.

Taking three batches of vitamin B for process verification₆The injection and reference preparation (batch No. 6022372, available from Phersusuki) were prepared by respectively adopting self-constructed analysis method (chromatographic conditions and impurity enriched solution analysis method) and Chinese pharmacopoeia method for vitamin B₆The injection is used for detecting related substances, the preparation method of the system applicability solution, the test solution and the control solution is the same as that of example 5, and the results are shown in the following table:

according to the results, the analysis method developed by the invention is obviously superior to the second vitamin B in the 2020 edition of Chinese pharmacopoeia in the detected number and the detected amount of impurities₆Method for detecting related substances of injection.

Typical spectra of reference formulations monitored for related substances in the process comparisons are shown in FIGS. 28-31.

It should be noted that the numerical values or numerical end points referred to in the technical solutions of the present invention are not limited to the numbers per se, and those skilled in the art can understand that they include allowable error ranges that are widely accepted in the art, such as experimental error, measurement error, statistical error, random error, and the like, and the error ranges are included in the scope of the present invention.

The foregoing examples and description are presented to enable others skilled in the art to make and use the invention, and are provided to enable others skilled in the art to make and use the invention with various modifications as are suited to the particular use contemplated. Therefore, the present invention includes but is not limited to the above embodiments, and modifications and improvements made by others skilled in the art without departing from the scope of the present invention are within the protection scope of the present invention based on the contents provided by the present invention.

Claims

1. Vitamin B₆A process for the preparation of an impurity compound, characterized by comprising the steps of:

step 1: preparation of vitamin B₆An enriched mother liquor of impurity compounds;

step 2: detection of vitamin B Using HPLC method₆The formation of impurity compounds;

and step 3: separating the enriched mother liquor obtained in the step (1) by adopting a preparative chromatographic column which takes octadecylsilane chemically bonded silica with polar groups bonded on the surface of the silica as a filler, and simultaneously carrying out qualitative and purity analysis by using a liquid chromatography-mass spectrometer;

and 4, step 4: merging and collecting the high-purity target fractions obtained in the step 3;

and 5: decompressing the fraction collected in the step 4 to obtain vitamin B₆A salt-containing product of impurity compounds;

step 6: desalting the salt-containing product by preparative liquid chromatography, and combining all collected target fractions;

and 7: concentrating the target fraction obtained in step 6 under reduced pressure to obtain vitamin B₆An impurity compound;

、

or

。

2. Vitamin B₆A process for the preparation of an impurity compound, characterized by comprising the steps of:

step 2: detection of vitamin B Using HPLC method₆Formation of impurity compounds;

and 5: carrying out reduced pressure distillation on the fraction collected in the step 4 to obtain vitamin B₆An impurity compound;

wherein the vitamin B₆The impurity compounds are:

。

3. the method according to claim 1 or 2, wherein vitamin B is prepared in step 1₆The enrichment mother liquor of impurity compounds is carried out under the conditions of intensifying high temperature, illumination or oxidationIn (1).

4. The method of claim 3, wherein the elevated temperature is 75 ℃ to 100 ℃.

5. The method of claim 4, wherein the elevated temperature is 80 ℃.

6. The method according to claim 1 or 2, wherein the detection conditions of the HPLC method in step 2 are as follows: GL Sciences Inertsil ODS-3, 250mm × 4.6mm, 5 μm using octadecylsilane chemically bonded silica as filler; using 0.04% sodium pentane sulfonate solution with pH value adjusted to 2.8 by phosphoric acid as mobile phase A, using methanol as mobile phase B, and carrying out gradient elution; the flow rate was 1.0ml per minute; the detection wavelength is 210 nm; the column temperature is 20 ℃; sample introduction volume is 10 mul

。

7. The method of claim 1 or 2, wherein the chromatography column in step 3 is a Waters XBridge Prep Shield RP18, 5 μm, 30 x 150mm or fast separation column.

8. The method of claim 7, wherein the flash separation column is a 40g flash separation column or an 80g flash separation column.

9. The method according to claim 1 or 2, wherein the step of performing qualitative and purity analysis by the LC-MS in step 3 comprises the following steps: taking a proper amount of target fraction collected from a preparative chromatographic column, adding water for dilution to serve as a test solution, taking 10 mu l of the test solution, injecting the test solution into a liquid chromatography-mass spectrometer, and taking octadecylsilane chemically bonded silica as a filler; taking 0.01mol/L ammonium formate solution with pH value adjusted to 3.0 by formic acid as a mobile phase A, taking methanol as a mobile phase B, and carrying out gradient elution; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; electrospray positive ion mode (ESI +),

。

10. the method according to claim 1 or 2, wherein the high purity target fraction in the step 4 is a target fraction having a purity of not less than 95%.

11. The method according to claim 1, wherein the reduced pressure treatment in the step 5 is concentration under reduced pressure or distillation under reduced pressure.

12. The method according to claim 1, wherein the preparative liquid chromatography in step 6 is a rapid separation column using octadecylsilane bonded silica gel of 20-35 μm as a packing or a Waters XBridge Prep Shield RP18 of 5 μm and 30 x 150mm, and water and methanol or acetonitrile are used as mobile phases, and the flow rate is set to 20-25 ml/min.

13. The method according to claim 12, wherein the mobile phase is water and methanol, and the flow rate is set to 20 ml/min.

14. The method according to claim 6, wherein the mobile phase aqueous phase adopted by the chromatographic column in the step 3 is an aqueous solution of buffer salt or ion-pair reagent.

15. The method of claim 14, wherein the buffer salt comprises formic acid or formate, trifluoroacetic acid, acetic acid or acetate, phosphoric acid or phosphate, citric acid or citrate, and wherein the salt comprises potassium, sodium, or ammonium salt.

17. The method of claim 16, wherein the alkyl sulfonate is heptane sulfonate, pentane sulfonate, or octane sulfonate.

18. The method according to claim 14, wherein the flow rate of the chromatographic column in the step 3 is 10-30 ml/min.

19. The method as claimed in claim 18, wherein the flow rate of the chromatographic column in the step 3 is 15-25 ml/min.

20. The method of claim 19, wherein the chromatography column of step 3 is used at a flow rate of 15ml/min to 20 ml/min.

21. The method of claim 20, wherein the mobile phase aqueous phase used in the chromatographic column of step 3 is an ammonium formate solution.

22. The method as claimed in claim 17, wherein the mobile phase of the chromatographic column in step 3 is sodium pentanesulfonate solution.

23. The method of claim 21, wherein the ammonium formate is present in a concentration of 1mM to 500 mM.

24. The method of claim 23, wherein the ammonium formate is present in a concentration of 10mM to 50 mM.

25. The method of claim 24, wherein the ammonium formate is present in a concentration of 10 mM.

26. The method of claim 6, wherein the chromatographic column in step 3 is Waters Xbridge Prep Shield RP18, 5 μm,30X 150mM, mobile phase 10mM ammonium formate solution, wherein said vitamin B₆The impurity compounds are:

or

。

27. The method as claimed in claim 6, wherein the chromatographic column in step 3 is a rapid separation column, the mobile phase A is 0.04% sodium pentane sulfonate solution, the phase B is methanol, and the gradient elution is carried out, the detection wavelength is 210nm and 290nm, and the flow rate is 20ml/min, wherein the vitamin B₆The impurity compounds are:

or

。

28. A compound of formula II in vitamin B₆The application of the product as an impurity reference substance in impurity inspection,

formula II.

29. A compound of formula III in vitamin B₆The application of the product as an impurity reference substance in impurity inspection,

and (3) a formula III.

30. A compound of formula IV in vitamin B₆The application of the product as an impurity reference substance in impurity inspection,

formula IV.

31. A compound of formula V in vitamin B₆The application of the product as an impurity reference substance in impurity inspection,

formula V.

32. Use according to any one of claims 28 to 31, wherein said vitamin B is₆The HPLC conditions for detecting the substances are as follows: chromatographic column using octadecylsilane chemically bonded silica as filler; using 0.04% sodium pentane sulfonate solution with pH value adjusted to 2.8 by phosphoric acid as mobile phase A, using methanol as mobile phase B, and carrying out gradient elution; the flow rate was 1.0ml per minute; the detection wavelength is 210 nm; the column temperature is 20 ℃; sample introduction volume is 10 mul

。

33. The use of claim 32, wherein the chromatography column is GL Sciences insetsil ODS-3, 250mm x 4.6mm, 5 μ ι η.

34. Vitamin B₆The detection method of related substances is characterized in that the detection method adopts high performance liquid chromatography to detect vitamin B₆And impurities in the preparation are detected, and the high performance liquid chromatography conditions comprise: chromatographic column using octadecylsilane chemically bonded silica as filler; with 0.04% pentane adjusted to a pH of 2.8 with phosphoric acidTaking sodium sulfonate solution as a mobile phase A and methanol as a mobile phase B, and performing gradient elution; the flow rate was 1.0ml per minute; the detection wavelength is 210 nm; the column temperature is 20 ℃; sample introduction volume is 10 mul

。

35. The method of claim 34, wherein the chromatography column is GL Sciences insetsil ODS-3, 250mm x 4.6mm, 5 μ ι η.